Alcoholism is characterized by excessive alcohol consumption, motivation to obtain alcohol, and relapse upon exposure to drug associated cues or stress. Biological mechanisms that maintain escalated alcohol consumption, and mechanisms that trigger relapse to alcohol seeking are therefore potential targets for development of novel medications. Rats and mice will consume alcohol e.g. in a two bottle free choice situation, and lever-press when alcohol is offered as a reinforcer. However, basal consumption and self-administration in these models may be unrelated to mechanisms that maintain alcoholism. They may instead be driven by taste preference, appetite for calories, or other factors unrelated to the addictive process. Our work therefore emphasizes evaluating candidate medications in models of escalated intake, such as animals selectively bred for high alcohol preference, or animals with a prolonged history of intermittent brain alcohol exposure. These different models may tap into different biological mechanisms, and are therefore complementary. Relapse-like behavior is studied in animals in which operant self-administration is first established, and then extinguished. It is well established that presentation of drug-associated cues, or exposure to stress will lead to resumption of level-pressing on the previously alcohol-delivering lever (reinstatement). Reinstatement induced by cue and stress, respectively, is differentially sensitive to pharmacological manipulations. Evaluating candidate drugs for their ability to suppress reinstatement induced by the two different types of stimuli provides information how treatments might be combined to obtain additive effects, and may also be valuable to guide optimal selection of target populations in initial clinical trials. Substance P and its NK1 receptor Blockade of NK1 receptors, the main receptor for substance P (SP) suppresses behavioral stress responses in experimental animals. NK1R mutants showed markedly decreased alcohol preference, and this predicted human activity in a surrogate marker trial (George et al 2008). In follow-up work, we have found that conditioned place preference for alcohol, as well as escalation of intake over time are also eliminated, in a gene-dose dependent manner, in the mutants. Furthermore, we have established that pharmacological blockade of NK1 receptors using L-703,606 suppresses alcohol consumption similar to genetic deletion of the receptor. Because L-703,606, which has been optimized for potency at the human NK1 receptor may have off-target effects in mice, we used the NK1R null-mutants to demonstrate that this suppression is indeed mediated by NK1Rs (Thorsell et al 2010). Because of the limitations of NK1 antagonists developed for human use as research tools in rats and mice, we have collaborated with Dr. Kenner Rice, and resynthesized a literature compound, L-822429 that has been engineered to possess high affinity for the rat NK1 receptor. Using this compound, we have found a potent, dose dependent suppression of stress-induced reinstatement of alcohol seeking by the antagonist, an effect thought to be predictive of clinical anti-relapse activity. In contrast, no activity is seen on reinstatement induced by alcohol-associated cues, suggesting the possibility of additive effects of an NK1R antagonist and opioid antagonists such as the FDA approved alcoholism medication naltrexone (Schank et al. in preparation). Corticotropin-Releasing Hormone (CRH) and its CRH1 receptor Both the elevated self-administration of alcohol and the increased behavioral sensitivity to stress in the post-dependent state is in large part mediated by an up-regulation of the CRH1 subtype of CRH receptors in the amygdala (Sommer et al. 2008). This converges with prior findings of a stress-sensitive / anxious behavioral phenotype in the msP rat, driven by an innate up-regulation of CRH1 receptors in the amygdala and several other brain regions in this line (Hansson et al. 2006). The elevated innate CRH1 expression of msP rats is rescued by voluntary alcohohol consumption (Hansson et al. 2007). We previously identified a series of CRH antagonists with suitable properties for clinical development (Gehlert et al. 2007;Thorsell et al, unpublished data). More recently, we have entered into an agreement with BMS, and evaluated another series of orally available, brain penetrant CRH1 antagonists to determine their suitability for use in forthcoming human trials (Thorsell et al, unpublished). These results have guided the decision to initiate an experimental medicine study in treatment seeking alcoholics in the NIAAA clinical program. Neuropeptide Y (NPY) NPY is a potent endogenous anti-stress compound and counteracts the behavioral stress effects of CRH. Activation of NPY-Y1 receptors, or blockade of presynaptic Y2 autoreceptors suppresses escalated post-dependent drinking, while leaving basal intake of alcohol in non-dependent animals unaffected (reviewed in Thorsell et al. 2006). Recently, we showed that NPY potently and dose-dependently suppresses reinstatement of alcohol seeking induced by the pharmacological stressor yohimbine (Cippitelli et al. 2010). This indicates that medications that drive the NPY system, either directly by activating Y1 receptors, or indirectly, would be potentially valuable both for suppression of excessive alcohol consumption and for relapse prevention. Orally available, brain penetrant Y1 agonists are currently unavailable. Under a CRADA with Johnson and Johnson Pharmaceuticals, we have therefore evaluated of a non-peptide compound targeting the Y2 receptor to determine its potential for clinical development. However, we found an inverted-U-shaped dose-response curve, indicating that at higher doses, blockade of Y2 heteroceptors on other, e.g. glutamatergic neurons, may counteract the effects of increased endogenous NPY release (Cippitelli et al, in press). A collaboration with Scripps Florida under a UO1 mechanism is underway to develop novel compounds with improved properties. Neuropeptide S (NPS) The NPS receptor is a recently deorphanized GPCR with effects on anxiety like-behavior. Initial work in collaboration with the Ciccocioppo laboratory indicates that NPS signaling may have a role in relapse to alcohol seeking (Canella et al. 2009). We have developed a screenable assay for the NPS receptor, and in collaboration with Drs Zheng and Austin of the NIHGR Chemical Genomics Center completed a screen of their library. A lead molecule has been optimized, and determined to be brain penetrant upon peripheral administration in rats, and to possess in vivo activity, in that pretreatment with the antagonist prevents NPS-induced suppression of feeding. A patent has been filed jointly with NHGRI (Liu et al. 2010;Marugan et al. 2010). Structural changes Alcohol remodels the brain, by inducing neurodegeneration, but also by modulating generation and survival of new neurons. We have shown that neurodegeneration induced by binge-like brain alcohol exposure in the entorhinal cortex is blocked by glutamatergic mGlu2/3 agonism. This effect is mediated by the growth factor TGF-beta, and leads to rescue of cognitive defects associated with binge-induced neurodegeneration (Cippitelli et al. 2010). Also, while alcohol-induced suppression of hippocampal neurogenesis is transient, subventricular zone effects are permanent, and reflect a reduction of the stem cell pool (Hansson et al. 2010). Exploratory projects A series of exploratory projects currently evaluates the role of long-term gene expression modulating mechanisms, such as DNA methylation, histone acetylation and micro-RNA, in induction and maintenance of escalated alcohol consumption and relapse vulnerability.